Studies on DNA Methylation and Gene Expression : With Special Reference to the Oncodevelopmental Protein Genes

In order to study the effect of DNA methylation on gene expression, human cancer cell lines were treated with 5-azacytidine that inhibited DNA methylation. Expression of the AFP, CEA and albumin genes were examined. In addition, a CEA probe was prepared to detect CEA mRNA and to define its size. The following results were obtained. 1） Human hepatoma cells, HuH-7, produce large quantities of AFP and albumin. Treatment of 5-azacytidine resulted in a reduction of hybridizable AFP and albumin mRNA levels at 3 to 4 weeks. This effect was temporary and the mRNA levels were restored to the original levels after 6 to 7 weeks of cultivation in the absence of 5-azacytidine. In contrast, 5-azacytidine treatment resulted in the induction of CEA mRNA at 2 to 4 weeks, but this mRNA was not detected after 6 to 7 weeks. 2） Human hepatoma cells, c-Hc-4, produce small quantities of AFP and albumin. Treatment of 5-azacytidine induced AFP and albumin mRNAs at 1 to 4 weeks. 3） In human colon cancer cells BM314, 5-azacytidine treatment reduced CEA, AFP and albumin mRNAs. 4） Southern blot analysis of DNAs from HuH-7, c-Hc-4 and Chang liver cells showed that the expression of the AFP and albumin genes were not correlated with gene hypomethylation as analyzed by Msp I and Hpa II digestion. 5） A CEA probe was prepared by extension of an oligonucleotide that corresponds to five amino acid residues at position 13 to 17 of CEA with reverse transcriptase. The hybridized mRNA size was 2.2kb by this probe using the Northern blot analysis. Since this size of mRNA is able to code a polypeptide of MW 72,000 and is considered compatible with CEA polypeptide, this probe may be useful for cloning the CEA gene.departmental bulletin pape